The studies proposed examine the regulation of the osteocalcin gene as a model for identifying DNA sequences and transcription factors that contribute to the osteoblast phenotype. Osteocalcin knock-out mice have increased bone formation, suggesting that silencers of osteocalcin gene transcription play an important role in the regulation of bone formation. Sequences in the rat osteocalcin gene have been identified that contribute to silencing of osteocalcin-CAT fusion genes. Sequences identical to the core motifs of these elements are found in several other osteoblast genes and these silencers are also induced in a prechondroblastic cell line (MLB13MYC clone 17 [C17]) that acquires an osteoblast-like phenotype in response to BMP-2. One of these silencers will be cloned and characterized along with other factors induced during BMP-directed cell differentiation. Using differential display PCR, four mRNAs that are induced post-BMP-2 treatment of C17 cells have been identified and the applicant has found that two of these products are expressed in a tissue-specific manner and represent novel sequences not currently found in currently available data bases. Full-length cDNAs encoding these products are to be isolated and their functions in developing bone determined. In addition, characterization of a sequence adjacent to the VDRE of the rat and human osteocalcin gene absolutely required for transcriptional activation by Vit D will be performed and isolation of proteins that interact with this DNA will be determined.